Standing wave interferometric microscope

ABSTRACT

A wide-field interferometric microscope comprising:
         A specimen holder, for holding a specimen at an analysis location;   An illuminator, for illuminating the specimen with input radiation, so as to cause it to emit fluorescence light;   A pair of projection systems, arranged at opposite sides of said analysis location, to collect at least a portion of said fluorescence light and direct a corresponding pair of light beams into a respective pair of inputs of an optical combining element, where they optically interfere;   A detector arrangement, for examining output light from said combining element,
 
wherein:
   The illuminator is configured to produce a standing wave of input radiation at the analysis location   The detector arrangement comprises exactly two interferometric detection branches.

The invention relates to a wide-field interferometric microscopecomprising:

-   -   A specimen holder, for holding a specimen at an analysis        location;    -   An illuminator, for illuminating the specimen with input        radiation, so as to cause it to emit fluorescence light;    -   A pair of projection systems, arranged at opposite sides of said        analysis location, to collect at least a portion of said        fluorescence light and direct a corresponding pair of light        beams into a respective pair of inputs of an optical combining        element, where they optically interfere;    -   A detector arrangement, for examining light output from said        combining element.

The invention also relates to a method of using such a microscope.

A microscope as set forth above is, for example, known from the field ofinterferometric Photo-Activated Localization Microscopy (iPALM), which,for example, is elucidated in more detail in the following references:

-   http:www.pnas.org/content/106/9/3125.full-   http:www.mechanobio.info/topics/methods/super-resolution-microscopy-intro    The iPALM technique can, in turn, be regarded as a refinement of the    conventional (non-interferometric) PALM technique, whereby the    former augments the latter with the ability to perform    resolution/image reconstruction axially as well as laterally.    This can be understood as follows:    -   In PALM, lateral super-resolution is achieved by sequentially        exciting spatially sparse subsets of objects (photoactivatable        fluorophores) in a specimen, causing temporal separation of        fluorescence emission from these different subsets. The        resolvability of objects within each of these sparse subsets is        greater than if the whole specimen were to be imaged in one go.        In essence, the resolution-limiting diffraction effects that one        would expect if one were to attempt to simultaneously image a        dense set of objects are circumvented by instead regarding the        set as a cumulative collective of sparse subsets, which are        sequentially imaged. The photoactivatable fluorophores are        caused to fluoresce in a two-step process, whereby:        -   In a preliminary step, a so-called “activating wavelength”            (or “activation wavelength”) is used to promote the            fluorophore from a non-emissive to an emissive state;        -   In a subsequent step, a so-called “exciting wavelength” (or            “excitation wavelength”) is used to cause radiative            “relaxation” of the activated fluorophore (fluorescence            excitation).            See, for example, the following reference for more            information on this process:-   http:/www.hindawi.com/journals/isrn/2012/619251/    -   In iPALM, the lateral (XY) super-resolution achieved in PALM is        taken a step further, by introducing a mechanism that will also        allow fine axial/depth (Z) resolution. This is achieved by        imaging the (fluorophores in the) specimen through a pair of        oppositely disposed projection systems (objective lenses,        optical columns), whose output beams are fed into an optical        combining element (specifically, a three-phase beam splitter),        where they optically interfere. The resulting interference        fringe pattern will be (very) sensitive to the axial (depth)        position of the object (fluorophore) being imaged, since this        will influence the relative path lengths of the interfering        beams. By using a detector arrangement comprising multiple        detectors (e.g. CCDs) to selectively look at phase-separated        outputs from the combining element, one can effectively        (mathematically) “translate” a given fringe pattern into a        deduced axial object position; in iPALM, three distinct outputs        from the combining element (mutually phase-shifted by 120°) are        observed using three different detectors (cameras), whereby the        relative intensities of the outputs observed by these cameras        will change in a predictable manner as a function of axial        fluorophore position.

Although iPALM is a useful technique, it does suffer from drawbacks.More specifically, it relies on a relatively complicatedoptical/detection architecture. In particular:

-   -   The employed three-phase beam splitter is an expensive and        fragile component that is difficult to manufacture. Its        performance is sensitive to temperature fluctuations and        mechanical vibrations, and it has a relatively long settling        time after being disturbed. Moreover, it is difficult to        optically align/adjust.    -   The employed three-phase beam splitter is also difficult to        mechanically scale up in size, e.g. to match cameras with a        larger field of view (without vignetting). Limiting factors in        this regard include tolerances on the planar optics of the beam        splitter, and coherence characteristics of the fluorescence        light.    -   The detection set-up requires the use of three        detectors/cameras, which increases bulk/decreases available        space, and increases expense.

For good order, it is noted that, in addition to PALM/iPALM, there arealso various other types of fluorescence microscopy in use, such asSTORM and dSTORM, for example. More information in this regard can, forexample, be gleaned from the following reference:

-   https://en.wikipedia.org/wiki/Super-resolution_microscopy

One should note the distinction between a wide-field microscope—whichcan be regarded as employing a planar imaging wave—and, for example, apoint scanning microscope (German: “Rastermikroscop”), which uses animaging beam that is focused to a point, and is thus (necessarily)scanned over an object to be imaged. The present invention relates tothe former (wide field). Examples of the latter (point scanning) are,for example, set forth in EP 0 491 289 A1 and the journal article by S.W. Hell et al., Enhancing the axial resolution in far-field lightmicroscopy: two-photon 4Pi confocal fluorescence microscopy, J. ModernOptics 41(4), pp. 675-681 (1994).

It is an object of the invention to address these issues. In particular,it is an object of the invention to provide an alternativedepth-resolved localization microscopy technique that utilizes aradically different illumination/detection configuration. Morespecifically, it is an object of the invention that does not require useof a three-phase beam splitter.

These and other objects are achieved in a microscope as specified in theopening paragraph above, characterized in that:

-   -   The illuminator comprises an optical cavity that is configured        to produce a standing wave of input radiation at the analysis        location;    -   The detector arrangement comprises exactly two interferometric        detection branches.

The following aspects of the invention merit explicit mention:

-   -   (i) The standing wave alluded to here may be produced using the        “activating” input light or the “exciting” input light, and will        extend in a direction along the (local) optical axis at the        analysis location.    -   (ii) This standing wave produces a (sinusoidal) modulation of        the input radiation that illuminates the specimen, and it has a        phase that can be tuned, e.g. by adjusting the “length” of the        optical cavity in which it is generated.    -   (iii) Aspect (ii) can be exploited to provide an alternative for        (at least) one of the three 120°-degree-phase-shifted beams used        in the conventional iPALM detection set-up; since three        detection beams are thus rendered unnecessary, one no longer        needs to use a troublesome three-phase beam splitter and        associated trio of cameras—instead, one can suffice with a        regular two-way beam splitter, which is much cheaper, less        fragile, and more easily manufactured (and scaled to larger        sizes).    -   (iv) The spatially modulated intensity distribution in the        standing wave admits innovative ways of activating/exciting the        fluorophores in the specimen, which can serve as a basis for new        effects and advantages.        These aspects of the invention will receive further elucidation        below.        Note that the invention is distinguished from:    -   Detector arrangements that use only one detector        (branch/channel), e.g. as set forth in US 2005/0006597 A1 and EP        0 491 289 A1. In such set-ups, although one can observe an        interferometric image, one cannot meaningfully interpret        detected intensities; for example, one does not know if an        above-average intensity value is due to constructive        interference effects, or instead due to a fluorophore with a        relatively high emission rate, or to a combination of both.        Using more than one channel allows examination of intensity        ratios, thus mitigating this problem.    -   Detector arrangements that use three detectors        (branches/channels), e.g. as described above/below, and as set        forth in US 2006/0291043 A1 (in which it should be noted that no        interferometric imaging is done: the three employed cameras are        only used to detect different wavelengths).

There are various ways in which to realize/configure an illuminator ofthe type used by the invention. In a particular embodiment, theilluminator comprises:

-   -   A beam splitter, to produce a pair of coherent beams from a        single source (e.g. a laser);    -   A pair of reflectors, to direct each of said pair of coherent        beams through a respective one of the employed pair of        projection systems,        whereby said optical cavity comprises said beam splitter and        said pair of reflectors. An example of such a set-up is depicted        in FIG. 1, for instance. Such a configuration can be regarded as        a “dual-insertion” architecture, because the standing wave is        generated using two, oppositely directed input beams.

In an alternative embodiment to that set forth in the previousparagraph, the illuminator comprises:

-   -   A laser, located at a first side of said analysis location, to        direct an input beam along a common optical axis of said pair of        projection systems and through said specimen in a first        direction;    -   A movable mirror, located at a second, opposite side of said        analysis location and arranged normal to said common optical        axis, to reflect said input beam back upon itself and through        said specimen in a second, opposite direction.        An example of such a set-up is depicted in FIG. 2, for instance.        Such a configuration can be regarded as a “single-insertion”        architecture, because the standing wave is generated using a        single input beam, which, however, is reflected back upon itself        by the employed movable mirror; in this case, a standing wave        cavity is formed by said movable mirror and the lasing cavity in        the laser. In general, the movable mirror will have associated        collimation optics.

In a refinement of the set-up described in the previous paragraph, theilluminator can optionally comprise an optical diode or 50:50 plate beamsplitter (for example) provided between said laser and said movablemirror. Such an embodiment serves to mitigate feedback effects in thelasing cavity.

In the single-insertion embodiments just discussed, adjusting the axialposition of the movable mirror (along the local optical axis) allows thephase of the standing wave (at the analysis location) to be modified. Asimilar effect can be achieved in the preceding “dual-insertion”embodiment by, for example:

-   -   Incorporating an adjustable optical retarder element in the path        of at least one of the two input beams (as shown in FIGS. 1 and        3); or/and    -   Moving (at least) one of the pair of reflectors (and, if        necessary, co-moving the beam splitter), so as to adjust the        axial separation of the reflectors.

As already stated above, the standing wave utilized in the presentinvention may be generated using light from the activating light source(e.g. a laser with a wavelength of 405 nm) or the exciting light source(e.g. a laser with a wavelength of 488 nm, 561 nm, 639 nm or 750 nm);such aspects relate to the illumination architecture of the inventivemicroscope. In addition to illumination optics, the invention is alsoconcerned with the detection optics of the microscope. In that regard, aparticular embodiment of the invention is characterized in that theemployed optical combining element (OCE) comprises a two-waybeam-splitter (as already alluded to above). This can be used inconjunction with a detector arrangement comprising two cameras, whichlook at two mutually phase-shifted outputs from the (OCE): see FIG. 3,for example. In what follows, it will be explained why/how the standingwave generated in the present invention allows a less complicated OCEand a less complicated detector arrangement than in iPALM (and similartechniques).

Referring now to FIG. 4A, this shows a graph of measured intensity (Iin)versus axial position (Z) of (a fluorescing portion of) a specimen S asmeasured by detectors (cameras) Da (which registers intensity Iin1) andDb (which registers intensity Iin2) in a set-up such as that shown inFIG. 3 (whereby the suffix “in” denotes “interference”). It is notedthat Iin1 and Iin2 demonstrate a sinusoidal dependence on Z. Theintensity Iin on a given detector is determined by the sum/difference ofthe electromagnetic fields E_(B1) and E_(B2) associated with (travellingalong) beams B1 and B2, respectively, whereby:Iin1=(E _(B1) +EB2)² Iin2=(E _(B1) −EB2)².The emission beam path of the cavity produces a phase shift of π betweenIin1 and Iin2. The fluorescence wavelength in this particular instanceis 530 nm, and the associated period of the intensity signals Iin1(Z)and Iin2(Z) is thus 530 nm/4=132.5 nm; however, these particular valuesare not limiting upon the current discussion. In a correspondingfashion, FIG. 4B shows the so-called Normalized Differential Intensity(Qin) as a function of Z, whereby:Qin=(Iin1−Iin2)/(Iin1+Iin2).FIG. 4B is also sometimes referred to as a “calibration curve” for theemployed detector arrangement. It is noted that the slope of thiscalibration curve reduces significantly in zones such as r1 and r2,which respectively correspond to a local maximum and local minimum ofthe curve; in these “dud” zones r1, r2, there is therefore acorrespondingly lowered detection sensitivity. As a result, if, at/neara given Z-value, the value of Qin is extremal or near to extremal(corresponding to zones such as r1, r2), then it will be difficult toaccurately determine the Z-value in question, which is an undesirablesituation. This issue can be dealt with in different ways:

-   -   (a) In conventional iPALM, the underlying problem is addressed        by using three detection channels, which are mutually        phase-shifted by 120°/240°; as a result, if the Normalized        Differential Intensity (NDI) for a given Z-value and a given        pair of channels lands in a dud zone, then one can instead use        the NDI based on a different pair of channels, which (for the        same Z-value) will (necessarily) lie outside a dud zone.    -   (b) In contrast, the present invention does not need to rely on        such a third channel, and instead solves the problem of dud        zones in a completely different manner. In this regard,        reference is made to FIGS. 5A and 5B, which relate to the        innovative standing wave set-up of the current invention        (whereby the suffix “sw” denotes “standing wave”). In this        particular instance, the standing wave in question is generated        using an illumination wavelength of 488 nm, but that is not        limiting upon the current discussion. FIG. 5A shows the        intensity (Isw1) of a first standing wave as a function of axial        position (Z), and also shows the intensity (Isw2) of a second,        axially displaced standing wave as a function of axial position        (Z), whereby there is a phase difference Δφ=π between said first        and second standing waves. FIG. 5B shows the calibration curve        corresponding to FIG. 5A [Qsw versus Z, with        Qsw=(Isw1−Isw2)/(Isw1+Isw2)]. Note the “flank” zones r3, r4 in        which the slope of the curve is greatest, corresponding to        greatest sensitivity. By adding a phase shift δ to Isw1/Isw2        (e.g. by suitably moving retarding element R in FIG. 1 or 3),        one can cause the calibration curve of FIG. 5B—and, therefore,        the position of flank zones r3, r4—to shift along Z. In        particular, one can Z-shift the calibration curve of FIG. 5B so        that one of its flank zones (r3, r4; maximum sensitivity)        corresponds to a dud zone (r1, r2; minimum sensitivity) of the        calibration curve of FIG. 4B. In essence, one effectively takes        four measurements, namely:    -   Iin1, Iin2 at a first standing wave phase value Δφ;    -   Iin1′, Iin2′ at a second standing wave phase value δ+Δφ,        whereby the Quantum Efficiency (emission brightness) of the        observed fluorescing fluorophore(s) should not (significantly)        change during the measurement process (so that an observed        intensity change can be validly attributed to a standing wave        phase shift rather than a change in intrinsic brightness of the        fluorophore(s)); this will typically imply an exposure time of        the order of about 1-100 ms, for example. From these        measurements, the Z-position of an observed portion (fluorescing        fluorophore(s)) of the specimen can be determined. This can be        done by “fitting” the measured intensity values to reference        Q-versus-Z graphs obtained in a (previously performed)        calibration session in which intensity signals from a test        specimen (such as a gold nanoparticle) are registered as the        test specimen is deliberately moved along Z. Reference is made        to Embodiment 4 below for more information in this regard.

As already set forth above, the present invention uses an innovativeillumination set-up, which correspondingly allows an innovativedetection set-up to be employed. In a further aspect of the presentinvention:

-   -   Said standing wave is produced using said first type of        radiation;    -   Said selected fluorophores are activated in a depth region of        the specimen proximal to a local maximum of said standing wave.        Such a scenario exploits the fact that a standing wave generated        in the illuminator according to the invention will intrinsically        have localized maxima and minima extending axially through the        specimen, and that this effect can be exploited to activate        fluorophores in a depth region that is relatively thin relative        to a period of the standing wave.

In a particular aspect of the invention, wavefront modifying means areused to produce astigmatism in light entering the optical combiningelement. To this end, one could, for example, employ a cylindrical lensor mirror, or introduce a (cylindrical) stress into a planar mirror(such as a folding mirror), in at least one/preferably both of the dualdetection branches of the microscope. Introducing astigmatism (moregenerally: wavefront modification that varies in polarity as a functionof axial position) in this manner causes an associated Point SpreadFunction (PSF) to demonstrate ellipticity “oscillations” as a functionof Z—changing from elongate along Y, to circular, to elongate along X,etc. Observing the form of this PSF at a given axial position can thenbe used to help determine a Z-value for that position—more specifically,it acts as a check on the “sign” of a Z-coordinate of known amplitude.

For some further information on fluorophores and their use influorescence microscopy, reference is made to the following sources:

-   -   https://en.wikipedia.org/wiki/Fluorophore    -   J. Lippincott-Schwartz & G. Patterson, Photoactivatable        fluorescent proteins for diffraction-limited and        super-resolution imaging, Trends in Cell Biology 19 (11), pp.        555-565, Elsevier Ltd., 2009.

The invention will now be elucidated in more detail on the basis ofexemplary embodiments and the accompanying schematic drawings, in which:

FIG. 1 illustrates a longitudinal cross-sectional view of part of anembodiment of a microscope according to the present invention.

FIG. 2 illustrates a longitudinal cross-sectional view of part ofanother embodiment of a microscope according to the present invention.

FIG. 3 illustrates a longitudinal cross-sectional view of a particularembodiment of a microscope according to the present invention.

FIGS. 4A and 4B respectively show graphs of intensity (Iin) andNormalized Differential Intensity (Qin) as a function of axial position(Z) for two interfering light beams in an optical combining element.

FIGS. 5A and 5B respectively show graphs of intensity (Isw) andNormalized Differential Intensity (Qsw) as a function of axial position(Z) for two phase-shifted standing waves, produced in an illuminatoraccording to the current invention.

FIG. 6 illustrates a graph in which curves such as those in FIGS. 4B and5B have been combined/superimposed.

In the Figures, where pertinent, corresponding parts may be indicatedusing corresponding reference symbols.

EMBODIMENT 1

FIG. 1 illustrates a longitudinal cross-sectional view of part of anembodiment of a microscope (M) according to the present invention. Moreparticularly, it illustrates an embodiment of an illuminator IL for sucha microscope. In the Figure, a laser L produces a beam 1 of “inputradiation”, which, in the context of the present invention, may be anactivation beam or an excitation beam for respectivelyactivating/exciting a fluorophore in fluorescence microscopy. This beam1 serves to illuminate (activate and/or excite) (a collection offluorophores in) specimen S that is held on a specimen holder H at ananalysis location A, (ultimately) causing (part of) specimen S to emitfluorescence light. The analysis location A is straddled by a pair ofoppositely-located projection systems P1, P2, which will serve tocollect this fluorescence light and direct it onto a detectorarrangement D (to be discussed in the context of FIG. 3); for now, thepresent discussion will concentrate on the structure/functioning ofilluminator IL.

The beam 1 encounters a two-way beam splitter 3, which divides the beam1 into a pair of coherent light beams 5 a, 5 b, respectively located intwo different “branches” or “arms” that originate from a beam-splittingsurface 3′ in item 3. The beams 5 a, 5 b subsequently impinge on arespective pair of reflectors (e.g. mirrors) 7 a, 7 b, which divert thebeams 5 a, 5 b onto (or approximately onto) a common optical axis O ofco-linear projection systems P1, P2; in this way, diverted beam 5 atraverses analysis area A along O through P1, whereas diverted beam 5 btraverses analysis area A along O through P2, and these two divertedbeams produce a (longitudinal/axial) standing wave at location A (andelsewhere in the path/optical cavity A, 7 a, 3, 7 b, A). Asschematically illustrated in FIG. 3, such a standing wave 31 will havealternating maxima and minima disposed along axis Z (of illustratedCartesian coordinate system X, Y, Z), which extends parallel to O.

Also symbolically/generically shown are optics 9, 11, which, forexample, serve to focus/collimate the beams 5 a, 5 b. Moreover, as heredepicted, an adjustable retarding element R is located in one ofabovementioned “branches”, thus allowing the phase of the generatedstanding wave 31 to be adjusted. As an alternative or supplement tothis, one could also shift (at least) one of the reflectors 7 a, 7b—e.g. shift reflector 7 a as shown by the arrow symbol beside it.

EMBODIMENT 2

FIG. 2 illustrates a longitudinal cross-sectional view of part ofanother embodiment of a microscope according to the present invention;more particularly, it illustrates an embodiment of an illuminator IL forsuch a microscope. Certain parts of FIG. 2 that are also present in FIG.1 will not necessarily be discussed here; instead, the followingdiscussion will concentrate on the differences between the two Figures.

In FIG. 2, a canted mirror 17 (optional) is located at a first side(“P2-side” or “upstream”) of analysis location A; this is used to directan input beam from a laser L along common optical axis O of projectionsystems P2, P1 and through specimen S in a first direction (+Z). Use isalso made of a movable mirror (reflector) 13, which is arranged to besubstantially normal to optical axis O, can be displaced along O in acontrolled manner, and is situated at a second, opposite side (“P1-side”or “downstream”) of analysis location A; this serves to reflect saidinput beam back upon itself and through specimen S in a second, oppositedirection (−Z). The outgoing (+Z) and returning (−Z) beam from Linteract to produce a standing wave (inter alia at A). Displacement ofmirror 13 along axis O allows the phase of this standing wave to beadjusted. Also symbolically/generically shown are optics 19, 21, which,for example, serve a focusing/collimation function.

Optionally present in FIG. 2 is a device 15 such as an optical diode 15(e.g. a Faraday Isolator) or a 50:50 plate beam splitter.

EMBODIMENT 3

FIG. 3 illustrates a longitudinal cross-sectional view of a particularembodiment of a microscope M according to the present invention. Theillustrated microscope M comprises (inter alia) an illumination portion(to the right of axis O) and a detection portion (to the left of axisO). Said illumination portion essentially corresponds to the set-upshown in FIG. 1 (but could just as easily be based on the set-up shownin FIG. 2); therefore, so as to avoid unnecessary repetition, thefollowing discussion will concentrate on said detection portion.

As already set forth above, illumination of (a collection offluorophores in) specimen S—using suitably chosen activation andexcitation wavelengths—will cause (certain of) those fluorophores toemit fluorescence light, which is (partially) collected by projectionsystems P1, P2. Using canted dichroic mirrors (reflectors) 23, 25(positioned on axis O), light collected by P1 and P2 is respectivelydirected as beams B1, B2 into (a respective pair of input faces of)Optical Combining Element (OCE) C—which, in the current invention, canbe a (relatively simple) two-phase beam splitter (combiner) rather thana (more complicated) three-phase beam splitter (combiner); within OCE C,the beams B1 and B2 optically interfere and produce an interferencepattern (not depicted). A detector arrangement D—which here comprisestwo detectors Da, Db—is used to examine this interference pattern, bysimultaneously looking at it along two different (mutuallyphase-shifted) “channels”: see FIG. 4A, for example. Also symbolicallyshown in FIG. 3 are generic optics 27, 29, which, for example, serve afocusing/collimation function. Ideally, the beam-splitting surface C′ ofOCE C is located in the same plane as specimen S; in that case, thephases of fluorescence emission of the beams B1 and B2 are “balanced”relative to the beam splitter position.

EMBODIMENT 4

With reference to the elucidation already given above regarding FIGS.4A, 4B, 5A and 5B, a supplemental description will now be given as tohow an inventive microscope such as that depicted in FIG. 3 can be used.More particularly, the following discussion will concentrate on certainaspects of detection signal analysis/processing/interpretation.

FIG. 6 illustrates a graph in which curves such as those in FIGS. 4B and5B have been combined/superimposed. Because the component curves havedifferent frequencies (as a function of Z), they will inevitably crosseach other at certain points—such as in depicted zones r5, r6, forexample. In such zones, measurement sensitivity will tend to berelatively low.

This problem can be addressed using a technique that is also exploitedin iPALM. If the wavefront of the fluorescence light reaching the OCE Cis deliberately deformed so as to introduce astigmatism—e.g. bydeliberately mechanically stressing one/preferably both of the foldingmirrors 23, 25 in FIG. 3—then the associated Point Spread Function (PSF)33 will demonstrate ellipticity “oscillations” as a function ofZ—changing from elongate along Y, to circular, to elongate along X, etc.Observing the form of the PSF 33 at a given position can then be used todeduce a Z-value for that position. This is schematically depicted inFIG. 6, by illustrating exemplary PSF forms as a function of Z along theabscissa axis.

The basic mathematical analysis of the interference pattern in amicroscope according to the current invention is similar to thatpertaining to iPALM. For more information in this regard, reference is(for example) made to the mathematical discussion in U.S. Pat. No.7,924,432, which is incorporated herein by reference.

The invention claimed is:
 1. A wide-field interferometric microscope comprising: a specimen holder, for holding a specimen at an analysis location; an illuminator, for illuminating the specimen with input radiation, so as to cause it to emit fluorescence light; a pair of projection systems, arranged at opposite sides of said analysis location, to collect at least a portion of said fluorescence light and direct a corresponding pair of light beams into a respective pair of inputs of an optical combining element, where they optically interfere; wavefront modifying means for producing astigmatism in light entering the optical combining element; and a detector arrangement, for examining output light from said combining element, wherein: the illuminator comprises an optical cavity that is configured to produce a standing wave of input radiation at the analysis location; and the detector arrangement comprises exactly two interferometric detection branches.
 2. A microscope according to claim 1, wherein said illuminator comprises: a beam splitter, to produce a pair of coherent beams from a single source; and a pair of reflectors, to direct each of said pair of coherent beams through a respective one of said pair of projection systems, whereby said optical cavity comprises said beam splitter and said pair of reflectors.
 3. A microscope according to claim 2, wherein said illuminator comprises an adjustable optical retarding element arranged in a path of at least one of said coherent beams.
 4. A microscope according to claim 1, wherein said illuminator comprises: a laser, located at a first side of said analysis location, to direct an input beam along a common optical axis of said pair of projection systems and through said specimen in a first direction; and a movable mirror, located at a second, opposite side of said analysis location and arranged normal to said common optical axis, to reflect said input beam back upon itself and through said specimen in a second, opposite direction.
 5. A microscope according to claim 1, wherein said optical combining element comprises a two-way beam-splitter.
 6. A microscope according to claim 1, wherein said input radiation comprises: a first type of radiation, for activating selected fluorophores in the specimen; and a second type of radiation, for exciting a set of activated fluorophores, with the resultant emission of fluorescence light, and wherein said standing wave is produced using either said first type or said second type of radiation.
 7. A microscope according to claim 6, wherein: said standing wave is produced using said first type of radiation; and said selected fluorophores are activated in a depth region of the specimen proximal to a local maximum of said standing wave.
 8. A method of using a wide-field interferometric microscope comprising: a specimen holder, for holding a specimen at an analysis location; an illuminator, for illuminating a region of the specimen with input radiation, so as to cause at least one fluorophore in said region to emit fluorescence light; wavefront modification means; a pair of projection systems, arranged at opposite sides of said analysis location, to collect at least a portion of said fluorescence light and direct a corresponding pair of light beams into a respective pair of inputs of an optical combining element, where they optically interfere to produce an interference pattern; and a detector arrangement, for examining output light from said combining element, the method comprising: (I) using the illuminator to produce a standing wave of input radiation at the analysis location; (II) using the wavefront modification means to produce astigmatism in light entering the optical combining element; (III) using the detector arrangement to record an first intensity distribution of said interference pattern along exactly two different channels; (IV) altering a phase of said standing wave, and repeating step (II) for this phase-altered wave; and (V) using the intensity distributions recorded in steps (II) and (III) to derive an axial position of said fluorophore relative to a common optical axis of said pair of projection systems.
 9. A microscope according to claim 2, wherein said optical combining element comprises a two-way beam-splitter.
 10. A microscope according to claim 3, wherein said optical combining element comprises a two-way beam-splitter.
 11. A microscope according to claim 4, wherein said optical combining element comprises a two-way beam-splitter.
 12. A method of using a wide-field interferometric microscope, comprising: providing a specimen held on a specimen holder at an analysis location; using a wavefront modifying means to produce astigmatism in input radiation; producing a standing wave of the input radiation at the analysis location using an illuminator, the illumination causing at least one fluorophore in the analysis location to emit fluorescent light; collecting at least a portion of the fluorescent light using a pair of projection systems arranged at opposite sides of the analysis location; directing the corresponding pair of light beams collected by the pair of projection systems into a respective pair of inputs of an optical combining element, where they interfere to produce an interference pattern; recording an intensity distribution of the interference pattern along exactly two different channels; altering a phase of the standing wave, and recording an intensity distribution of the interference pattern along exactly two channels for the new interference pattern; and forming a representation of the specimen in which the axial position of the fluorophore relative to a common optical axis of the pair of projection systems is derived using the intensity distributions of the interference pattern recorded from the initial and altered phase of the standing wave. 